Liquid chromatographic assay for common sunscreen agents: application to in vivo assessment of skin penetration and systemic absorption in human volunteers

The purpose of the present study was to develop a reverse-phase high-performance liquid chromatographic (HPLC) assay for quantifying four common sunscreen agents, namely 2-hydroxy-4-methoxybenzophenone, 2-ethylhexyl-p-methoxycinnamate, 2-ethylhexylsalicylate (octylsalicylate) and salicylic acid 3,3,5-trimethcyclohexyl ester (homosalate) in a range of biological matrices. This assay was further applied to study the skin penetration and systemic absorption of sunscreen filters after topical application to human volunteers. Separation was achieved utilizing a Symmetry C(18) column with methanol-water as the mobile phase. The assay permits analysis of the sunscreen agents in biological fluids, including bovine serum albumin (BSA) solution, plasma and urine, and in human epidermis. The assay was linear (r2 > 0.99) with minimum detectable limits of 0.8 ng for oxybenzone, 0.3 ng for octylmethoxycinnamate, and 2 ng for homosalate and octylsalicylate. The inter- and intra-day variation for the four sunscreens was less than 3% at the upper end of the linear range and less than 6% at the lower end. Recoveries of sunscreens from plasma, 4% (w/v) BSA solution and epidermal membranes were within the range of 91-104%. Recoveries from urine of the four sunscreens, and oxybenzone with its metabolites were more than 86%. Up to approximately 1% of the applied dose of oxybenzone and its metabolites was detected in the urine. Appreciable amounts were also detected in the stratum corneum through tape stripping. The HPLC assay and extraction procedures developed are sensitive, simple, rapid, accurate and reproducible. Results from the preliminary clinical study demonstrate significant penetration of all sunscreen agents into the skin, and oxybenzone and metabolites across the skin.     

Production of protoporphyrin IX from 5-aminolevulinic acid and two of its esters in cells in vitro and tissues in vivo.

5-Aminolevulinic acid (ALA) and two of its esters were studied in cells in vitro and in vivo on skin of healthy hairless mice. In vitro, both esters, which are more lipophilic than ALA, induced higher PpIX fluorescence at lower concentrations compared with ALA. In vivo, ALA induced PpIX fluorescence more efficiently than the esters. The difference between ALA and the esters may be related to structures in the stratum corneum or to rate of penetration through this skin layer. The stratum corneum may bind the esters temporarily, and slow down their penetration into the living cells where PpIX is formed.     

9-fluorenylmethyl chloroformate as a fluorescence-labeling reagent for derivatization of carboxylic acid moiety of sodium valproate using liquid chromatography/tandem mass spectrometry for binding characterization: a human pharmacokinetic study

In High Performance Liquid Chromatographic (HPLC) determination of chemicals with acidic functions, different labeling agents are used to improve sensitivity of the assay. 9-Fluorenylmethyl chloroformate (FMOC-Cl), on the other hand, is a suitable labeling agent, which reacts with both primary and secondary amines and less readily with hydroxyl groups in alkaline conditions. However, the reagent has not been applied in labeling of chemicals with acidic function yet. In this study which is the first report on application of FMOC-Cl in derivatization and analysis of a drug with acidic function, valproic acid (VPA), one of a series of fatty carboxylic acids with anticonvulsant activity, was derivatized using the reagent and quantified in serum samples by HPLC with fluorescence detection. In addition, to document the reaction between the labeling agent and carboxylic acid moiety of the drug, we developed a liquid chromatography-tandem MS/MS (LC-MS/MS) method. Following liquid-liquid extraction, derivatization of the drug and an internal standard was achieved in alkaline medium. The elute was monitored by a fluorescence detector with respective excitation and emission wavelengths of 265 and 315 nm. The present method is more sensitive comparing with other published HPLC procedures for analysis of VPA. The assay is sensitive enough to measure drug levels obtained in human single dose studies with a limit of quantification of 0.01 μg/mL. Also the method is linear over the concentrations range of 0.01-32 μg/mL of VPA in human serum using 100 μL serum sample and 5 μL injection. The coefficient variation values of both inter and intra day analysis were less than 12% and the percentage error was less than 4%. The method performance was studied and the validated procedure applied in a randomized cross-over bioequivalence study of two different VPA preparations in 24 healthy volunteers.     

Bioanalysis and pharmacokinetics of chitosan ester in rabbit serum by HPLC with postcolumn fluorescence derivatization

Interest in antiatherosclerotic activity of chitosan ester (PS916) with a new form of sulfate amino polysaccharide derived from marine chitin has necessitated the development of a sensitive and specific method to study its pharmacokinetics. A sensitive and reproducible high-performance liquid chromatography (HPLC) with postcolumn fluorescence derivatization method was developed and validated for the determination of PS916 in rabbit serum. Chromatography was carried out using a C8 reversed-phase column with an isocratic mobile phase consisting of methanol-water (20:80, v/v) at a flow rate of 0.2 ml/min. The derivatization procedure involved postcolumn reaction with guanidine hydrochloride in an alkaline medium at 110 degrees C. The fluorometric detector was operated at 250 nm (excitation) and 435 nm (emission). The assay was linear over the concentration range of 5-100 microg/ml. The lower limit of detection (LLOD) was found to be 1.0 microg/ml. The proposed method was successfully applied for a pharmacokinetic study of PS916 in rabbits.     

渗透促进剂对紫草素透过大鼠腹部皮肤的作用

研究不同浓度的丙二醇、氮酮和油酸对紫草素透过大鼠腹部皮肤的影响．实验结果表明,不同浓度的丙二醇对紫草素经皮渗透均有促进渗透作用,丙二醇浓度为10％时促渗效果最佳,油酸也具有促进紫草素的经皮渗透作用,但是在本实验中氮酮抑制紫草素的经皮渗透。     

Analysis of cardiolipin in human muscle biopsy

Cardiolipin is a phospholipid that is specific to the inner mitochondrial membrane and essential for numerous mitochondrial functions. Accordingly, a quantitative assay for cardiolipin can be a valuable aspect of assessing mitochondrial content and functional capacity. The current study was undertaken to develop a simple and reliable method for direct analysis of the major molecular species of cardiolipin and with particular application for analysis of human skeletal muscle. The method that is presented is based on derivatization of cardiolipin in a total lipid extract with 1-pyrenyldiazomethane (PDAM), to form stable, fluorescent 1-pyrenylmethyl esters. The derivatization reaction takes 30 min on ice in a two-phase system (chloroform:methanol:H(2)O:H(2)SO(4)) containing 0.5-1.0mM PDAM and detergent. The contents of the major cardiolipin species in the derivatization mixture can be estimated by HPLC separation with fluorescent detection during a 20 min run on a reverse phase column and with HPLC grade ethanol/0.5mM H(3)PO(4) as the mobile phase. The recovery is about 80%. The method is specific and sensitive with quantitation limits of 0.5-1 pmol cardiolipin. The response of the fluorescence detector (peak area) is linear across a range 5-40 pmol. The assay is linear over the range between 0.3 and 3.0mg of tissue (R(2)=0.998). The assay provides good reproducibility and accuracy (within 5-10%).     

Light Fractionation Significantly Increases the Efficacy of Photodynamic Therapy Using BF-200 ALA in Normal Mouse Skin

Background

Light fractionation significantly increases the efficacy of 5-aminolevulinic acid (ALA) based photodynamic therapy (PDT) using the nano-emulsion based gel formulation BF-200. PDT using BF-200 ALA has recently been clinically approved and is under investigation in several phase III trials for the treatment of actinic keratosis. This study is the first to compare BF-200 ALA with ALA in preclinical models.

Results

In hairless mouse skin there is no difference in the temporal and spatial distribution of protoporphyrin IX determined by superficial imaging and fluorescence microscopy in frozen sections. In the skin-fold chamber model, BF-200 ALA leads to more PpIX fluorescence at depth in the skin compared to ALA suggesting an enhanced penetration of BF-200 ALA. Light fractionated PDT after BF-200 ALA application results in significantly more visual skin damage following PDT compared to a single illumination. Both ALA formulations show the same visual skin damage, rate of photobleaching and change in vascular volume immediately after PDT. Fluorescence immunohistochemical imaging shows loss of VE-cadherin in the vasculature at day 1 post PDT which is greater after BF-200 ALA compared to ALA and more profound after light fractionation compared to a single illumination.

Discussion

The present study illustrates the clinical potential of light fractionated PDT using BF-200 ALA for enhancing PDT efficacy in (pre-) malignant skin conditions such as basal cell carcinoma and vulval intraepithelial neoplasia and its application in other lesion such as cervical intraepithelial neoplasia and oral squamous cell carcinoma where current approaches have limited efficacy.     

Determination of amlodipine in human plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and specific HPLC method has been developed for the assay of amlodipine in human plasma. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl), solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Nortriptyline hydrochloride was used as an internal standard. The assay was linear over the concentration range of 0.25–18.00 ng/ml. Both of the within-day and day-to-day reproducibility and accuracy were less than 11.80% and 12.00%, respectively. The plasma profile following a single administration of 10 mg amlodipine to a healthy volunteer was presented.     

The effect of folic acid on porphyrin synthesis in tumors and normal skin of mice treated with 5-aminolevulinic acid or methyl 5-aminolevulinate.

5-Aminolevulinic acid (ALA) or its derivative methyl 5-aminolevulinate (MAL) combined with folic acid was applied in nude mice bearing human colon adenocarcinoma. The aim of the study is to see whether folic acid may increase biosynthesis of porphyrins in tumor tissue after systemic or topical administration of ALA or MAL. The production of porphyrins was determined by spectrofluorometric measurements with an optical fibre probe. It was found that the porphyrin production after i.p injection of 200 mg kg(-1) ALA or MAL was significantly increased by i.p injection of 100 mg kg(-1) folic acid. However, in the case of topically applied 20% ALA, folic acid had no effect. In the case of topically applied 20% MAL, folic acid (i.p or topically applied) reduced the porphyrin synthesis. This might be used for the protection of normal skin against photosensitization. The effects of folic acid were similar in tumors and normal skin. Two mechanisms may explain the results: enhancement of the efficiency of the rate-limiting enzyme porphobilinogen deaminase by folic acid or interference of folic acid with the transport of ALA and MAL to and into the cells synthesizing porphyrins in the tissues. The present data seem to favour the latter mechanism. Folic acid may have a role as an adjuvant in photodynamic therapy with systemically administered ALA and its derivatives.     

几种辛香中药对辣椒碱巴布剂促透皮吸收作用的研究

通过对辛香中药的研究筛选辣椒碱巴布剂的透皮吸收促进剂,提高辣椒碱的透皮扩散速率。采用分别在辣椒碱巴布剂中添加薄荷醇、冰片、桉叶油3种辛香类中药的方法,以月桂氮酮为对照,进行透皮渗透试验。结果表明3种辛香类中药和月桂氮酮对药物的迁移都有一定的促进作用,其中以桉叶油最为突出,其次为冰片、薄荷、氮酮。     

A Preformulation Strategy for the Selection of Penetration Enhancers for a Transungual Formulation

Onychomycosis is associated with the cutaneous fungal infection of the nail and the nail folds (skin surrounding the nail). It is therefore important to target drug delivery into the nail folds along with nail plate and the nail bed. Systematic and strategic selection of the penetration enhancers specific for the skin and the nail is discussed. Twelve penetration enhancers were screened for their ability to improve solubility, in vitro nail penetration, in vitro skin permeation, and in vitro skin penetration of the antifungal drug ciclopirox olamine. In contrast to transdermal drug delivery, the main selection criteria for skin penetration enhancer in topical drug delivery were increased drug accumulation in the epidermis and minimal permeation across the skin. Thiourea improved the solubility and nail penetration of ciclopirox olamine. It also showed enhancement in the transungual diffusion of the drug. Propylene glycol showed a 12-fold increase in solubility and 3-fold increase in epidermal accumulation of ciclopirox olamine, while minimizing the transdermal movement of the drug. Thiourea was the selected nail permeation enhancer and propylene glycol was the selected skin penetration enhancer of ciclopirox olamine. A combination of the selected enhancers was also explored for its effect on drug delivery to the nail and nail folds. The enhancer combination reduced the penetration of ciclopirox in the skin and also the permeation through the nail. The proposed preformulation strategy helps to select appropriate enhancers for optimum topical delivery and paves way towards an efficient topical formulation for passive transungual drug delivery.     

Simultaneous determination of the enantiomers of esmolol and its acid metabolite in human plasma by reversed phase liquid chromatography with solid-phase extraction

A stereoselective RP-high performance liquid chromatography (HPLC) assay to determine simultaneously the enantiomers of esmolol and its acid metabolite in human plasma was developed. The method involved a solid-phase extraction and a reversed-phase chromatographic separation with UV detection (lambda = 224 nm) after chiral derivatization. 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate (GITC) was employed as a pre-column chiral derivatization reagent. The assay was linear from 0.09 to 8.0 microg/ml for each enantiomer of esmolol and 0.07-8.0 microg/ml for each enantiomer of the acid metabolite. The absolute recoveries for all enantiomers were >73%. The intra- and inter-day variations were <15%. The validated method was applied to quantify the enantiomers of esmolol and its metabolite in human plasma for hydrolysis studies.     

Determination of arginine-vasotocin and isotocin in fish plasma with solid-phase extraction and fluorescence derivatization followed by high-performance liquid chromatography

A new HPLC assay for plasma arginine-vasotocin (AVT) and isotocin (IT) determination based on fluorescence detection preceded by combination of solid-phase extraction (SPE) and fluorescence derivatization is presented. Plasma samples retained on solid support were purified and then derivatized by the fluorescent compound 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The peptide derivatives were eluted from cartridges, pre-concentrated and analyzed by HPLC system with fluorescent detection. The separation was carried out on a reversed-phase column with solvent gradient system. The assay was linear in the range 15-220 pmol ml(-1) for AVT r2=0.998 and 10-220 pmol ml(-1) for IT r2=0.996. The detection limits for AVT and IT were 0.8 and 0.5 pmol ml(-1) (3:1, signal-to-noise), respectively. The recoveries of derivatized hormones were in the range 89-93%. Both of the inter- and intra-day assay precision were below 5.5 and 9% for AVT and IT, respectively. The assay should be also applicable to plasma and tissue samples from other animals with only minor modification.     

Physicochemical and Safety Evaluation of 5-Aminolevulinic Acid in Novel Liposomes as Carrier for Skin Delivery

In this study we successfully entrapped 5-aminolevulinic acid (ALA) in liposome, although it exists as a zwitter ion. A molar ratio of 2:1:2.5 phosphatidyle-thanolamine (PE)/cholesterol/sodium stearate represented the best condition to achieve high entrapment efficiency (29.37 ± 1.21%), and the average vehicle size was 133.6 ± 2.8 nm. After 32 days of storage, the vehicle sizes of formulations with PE series were still approximately less than 200 nm. The safety of liposomes was tested and ensured both with regard to cellular cytotoxicity and erythrocyte hemolysis. Safety studies showed that liposome formulations did not affect cell viability except when both potassium stearate and sodium oleate were added. Moreover, PE and PE/cholesterol did not damage human erythrocytes in this study. The range of the hemolytic effect caused by liposomes was 5 to 37% and the effect was dependent on the amount of sodium stearate added to the formulation. According to the release rates and skin penetration of ALA liposomes in vitro, PE/cholesterol/sodium stearate liposomes might increase skin penetration, and it was shown that penetration across the stratum–corneum (sc) layer was the rate-limiting process. Images from confocal laser scanning microscopy (CLSM) confirmed the great potency of liposomes for delivering ALA into skin.     

Fluidity enhancement: a critical factor for performance of liposomal transdermal drug delivery system

Liposomes are well known lipid carriers for drug delivery of bioactive molecules encapsulated inside their membrane. Liposomes as skin drug delivery systems were initially promoted primarily for localized effects with minimal systemic delivery. Subsequently, a novel vesicular system, transferosomes was reported for transdermal delivery with efficiency similar to subcutaneous injection. The multiple bilayered organizations of lipids applied in these vesicles structure are somewhat similar to complex nature of stratum corneal intercellular lipids domains. The incorporation of novel agents into these lipid vesicles results in the loss of entrapped markers but it is similar to fluidization of stratum corneum lipids on treatment with a penetration enhancer. This approach generated the utility of penetration enhancers/fluidizing agents in lipids vesicular systems for skin delivery. For the transdermal and topical applications of liposomes, fluidity of bilayer lipid membrane is rate limiting which governs the permeation. This article critically reviews the relevance of using different types of vesicles as a model for skin in permeation enhancement studies. This study has also been designed to encompass all enhancement measurements and analytical tools for characterization of permeability in liposomal vesicular system.     

Physicochemical and safety evaluation of 5-aminolevulinic acid in novel liposomes as carrier for skin delivery

In this study we successfully entrapped 5-aminolevulinic acid (ALA) in liposome, although it exists as a zwitter ion. A molar ratio of 2:1:2.5 phosphatidyle-thanolamine (PE)/cholesterol/sodium stearate represented the best condition to achieve high entrapment efficiency (29.37 +/- 1.21%), and the average vehicle size was 133.6 +/- 2.8 nm. After 32 days of storage, the vehicle sizes of formulations with PE series were still approximately less than 200 nm. The safety of liposomes was tested and ensured both with regard to cellular cytotoxicity and erythrocyte hemolysis. Safety studies showed that liposome formulations did not affect cell viability except when both potassium stearate and sodium oleate were added. Moreover, PE and PE/cholesterol did not damage human erythrocytes in this study. The range of the hemolytic effect caused by liposomes was 5 to 37% and the effect was dependent on the amount of sodium stearate added to the formulation. According to the release rates and skin penetration of ALA liposomes in vitro, PE/cholesterol/sodium stearate liposomes might increase skin penetration, and it was shown that penetration across the stratum-corneum (sc) layer was the rate-limiting process. Images from confocal laser scanning microscopy (CLSM) confirmed the great potency of liposomes for delivering ALA into skin.     

Enhancer aided in vitro permeation of atenolol and prazosin hydrochloride through mice skin

Effect of penetration enhancers were studied on the permeation of antihypertensive drugs prazosin hydrochloride and atenolol through full thickness skin of swiss albino mice. Atenolol was delivered to skin from saturated alcoholic solution containing 5% of 1-decanol and alcohol alone, while prazosin hydrochloride was saturated in dimethyl formamide(DMF, 5% v/v in water) and dimethyl sulfoxide(DMSO, 5% v/v in water). Atenolol permeation was augmented significantly in decanolic solution and also in pure alcohol. In case of prazosin hydrochloride, significant enhancement of permeation was shown by DMSO but not by DMF.     

Diffusion Behaviors of Fluorescence Probe Molecules Through the Stratum Corneum Layer Under Physical Stress

This study experimentally demonstrates how application of an external physical stress onto the skin membrane affects the permeation of penetrating molecules. As a proxy of active compounds, in this study, a series of fluorescence probe molecules were utilized. We observed that skin permeation could be enhanced by imparting vertical strokes from a tapping head consisting of projections onto the skin. This was confirmed with consistency from in vitro and in vivo transdermal permeation studies. After an effective physical stress was applied to the skin, the permeation depth of probe molecules remarkably increased, which was comparable to the case of topical treatment. This seems to arise from temporal disordering of the stratum corneum layer in response to the applied physical stress.     

Determination of amphetamine in human urine by dansyl derivatization and high-performance liquid chromatography with fluorescence detection

A simple procedure for the determination of amphetamine in urine with minimal sample preparation is described. This method involves direct addition of human urine to an acetone-dansyl chloride solution for simultaneous deproteinization and fluorescence derivatization. The derivatized amphetamine is then measured by HPLC with fluorescence detection. It eliminates the extraction procedures often required by other HPLC or GC methods. The effects of pH, temperature and reaction time on the derivatization reaction were investigated. The stability of amphetamine-dansyl chloride in different storage conditions was examined. The detection limit and linearity associated with this assay are discussed.